Gene expression divergence during Drosophila head development on single cell resolution

了解地球上观察到的生命形式多样性的起源所涉及的过程是生物学研究的一个主要目的。成体形态是在胚胎和胚胎后发育过程中通过发育基因产物的作用而定义的，这些基因产物组织在基因调控网络(GRN)中。基因表达的自然变异以及发育基因的调控已被证明是形态多样化的主要驱动力。虽然相对简单性状差异的发育基础正在被揭示，但迄今为止，对导致复杂性状形态差异的分子变化缺乏全面的理解。为了缩小这一差距，我们研究了三个密切相关的果蝇物种头部发育的进化。昆虫头部是一种复杂的结构，由感知环境信息的主要感觉器官(如复眼和触角)和处理信息以触发行为反应的大脑组成。果蝇的头部由一个复合眼-触角成像盘发育而来，在幼虫阶段经历主要的图案形成、生长和分化过程。我们和其他人之前已经量化了三种果蝇物种黑腹果蝇、拟果蝇和毛果蝇在成虫头部大小和形状上的差异。为了确定与观察到的变异相关的候选基因，我们通过对整个发育过程中的整个眼睛触角成像盘进行测序(即Bulk-RNAseq)，生成了一个全面的比较转录数据集。基于这一数据，我们揭示了高水平的上下文相关的基因表达和调控，概括了发育中的眼睛-触角想象盘的合成性质。然而，由于Bulk-RNAseq数据不能提供足够的组织特异性分辨率，明确的候选基因和发育过程的识别受到阻碍。因此，我们建议在单细胞水平上利用单细胞信使核糖核酸序列(ScRNAseq)来研究基因表达的动态和差异。我们将建立黑腹大鼠、拟拟大鼠和毛氏大鼠眼触角成像盘发育的五个阶段的单细胞表达图谱。这些数据将被用来揭示关键的发育基因和代表发育头部不同区域的特定细胞团中的调控相互作用。观察到的细胞团在基因表达和GRN结构方面的种间比较将有助于识别不同的细胞群体。这些细胞群中的两个将应用CRISPR/Cas9介导的基因组编辑和集群特定细胞的重新测序进行功能验证。通过这个拟议的项目，我们将以前所未有的分辨率深入了解果蝇头部的发育和进化。此外，我们将有助于对上下文相关基因表达和调控的更广泛的机制理解。

Understanding the processes involved in the origin of the diversity of life forms observed on earth is a major aim of biological research. Adult morphology is defined during embryonic and postembryonic development by the action of developmental gene products, which are organized in gene regulatory networks (GRNs). Natural variation in the expression and thus the regulation of developmental genes has been shown to be a main driver for morphological diversification. While the developmental underpinnings of differences in relatively simple traits are being revealed, a comprehensive understanding of the molecular changes responsible for morphological divergence of complex traits is missing to date. To close this gap, we study the evolution of head development in three closely related Drosophila species. The insect head is a complex structure that consists of the main sensory organs (e.g. the compound eye and antennae) to perceive environmental information and the brain to process information to trigger behavioral responses. The Drosophila head develops from a composite eye-antennal imaginal disc that undergoes major patterning, growth and differentiation processes during larval stages. We and others have previously quantified differences in adult head size and shape among the three Drosophila species D. melanogaster, D. simulans and D. mauritiana. To identify candidate genes involved in the observed variation, we generated a comprehensive comparative transcriptomic dataset by sequencing entire eye-antennal imaginal discs throughout development (i.e. bulk-RNAseq). Based on this data we revealed a high level of context dependent gene expression and regulation recapitulating the composite nature of the developing eye-antennal imaginal discs. The identification of clear candidate genes and developmental processes, however, is hampered because the bulk-RNAseq data does not provide sufficient tissue specific resolution. Therefore, we propose here to study gene expression dynamics and divergence on single cell level using single cell mRNA sequencing (scRNAseq).We will establish a single cell expression atlas for five stages of eye-antennal imaginal disc development for D. melanogaster, D. simulans and D. mauritiana. This data will be used to reveal key developmental genes and regulatory interactions in specific cell clusters representing different regions of the developing head. The interspecific comparison of observed cell clusters with respect to gene expression and GRN architecture will allow identifying diverging cell populations. Two of these cell clusters will be functionally validated applying CRISPR/Cas9 mediated genome editing and re-sequencing of cluster specific cells. With the proposed project we will gain major insights into the development and evolution of the Drosophila head on an unprecedented resolution. Additionally, we will contribute to a broader mechanistic understanding of context dependent gene expression and regulation.